Physiologically active substances, such as hormones and enzymes, which play important roles in life activity, are proteins (polypeptides), and the high order structure is important in maintaining the activity. A disulfide bond that is formed between two cysteines is a component forming a high order structure. For example, insulin is secreted from B cells in the islets of Langerhans in the pancreas and is the most important hormone in storing or using sugar, amino acid and fatty acid, and in maintaining homeostasis of blood glucose levels. Insulin is a polypeptide that consists of an A chain comprising 21 amino acids and a B chain comprising 30 amino acids, and has one disulfide bond within the A chain and two disulfide bonds between the A chain and the B chain. When these 3 disulfide bonds are formed inaccurately, insulin will have no activity. Disulfide bonds are thought to be formed by enzymes, such as thioredoxin and protein disulfide isomerase, under an oxidation-reduction environment formed by glutathione and the like in a process where translated proteins are secreted to the endoplasmic reticulum and then transferred to the Golgi complex (Hwang, C. et al., Science 257: 1496–1502, (1992)., Gething, M. & Sambrook, J., Nature 355: 33–45, (1992), Bardwell, J. C. A. & Beckwith, J., Cell 74: 769–771, (1993)). It has been shown that these enzymes act to form disulfide bonds in in vitro experiments where the disulfide bond formation of proteins denatured to have no disulfide bond is enhanced when they are placed under reduction conditions and under the presence of the enzymes (Lyles, M. M. & Gilbert, H. F., Biochemistry 30: 613–619, (1991)., Lyles, M. M. & Gilbert, H. F., Biochemistry 30: 619–625, (1991)., Pigiet, V. F. & Schuster, B. J. Proc. Natl. Acad. Sci. USA 83:7643–7647, (1986)). Further, protein disulfide isomerase is known to have an effect of rearranging incorrect disulfide bonds into correct disulfide bonds (Weissman, J. S. & Kim, P. S, Nature 365:185–188, (1993)., Laboissiere, M. C. A. et al., J. Biol. Chem. 270: 28006–28009, (1995)).
Some physiologically active polypeptides that are present only in trace amounts in vivo, such as insulin, glucagon, interferon, calcitonin and growth hormone, are now being produced using established gene recombination techniques in large amounts as gene-recombinant proteins in prokaryotes and eukaryotes. In particular, an expression system in prokaryotes is widely used because of the large amount produced and the low production costs. However, among expression systems of prokaryotes, particularly a system in which proteins are expressed as inclusions within the cells of Escherichia coli has the advantage of a large amount of expression, but such a system cannot provide an environment for disulfide bond formation. Hence, proteins having disulfide bonds expressed in this expression system must be denatured once after isolation and purification, and then an environment for disulfide bond formation must be established. These steps are complex and may increase the cost. In contrast, a system in which proteins are expressed in the periplasm of Escherichia coli is an environment which enables disulfide bond formation therewithin, but the expression amount is limited. Moreover, a yeast expression system enables disulfide bond formation within the cells before secretion, but its expression amount is low.
A recently developed expression system of gene-recombinant protein using Bacillus brevis (Bacillus brevis; referred to as bacteria of the genus Brevibacillus according to a new classification) has received attention as a mass production system of gene-recombinant proteins with disulfide bonds, because polypeptides having disulfide bonds (human epidermal growth factor and the like) in an active state, that is, having accurately formed disulfide bonds are secreted and expressed in large amounts in media (Japanese Patent No. 2082727, Japanese Patent Application Laying-Open (Kokai) No. 62-201583, Konishi, H. et al., Appl. Microbiol. Biotechnol., 34:297–302 (1990), Shigezo UDAKA, Journal of Japan Society for Bioscience, Biotechnology and Agrochemistry 61, 669–676 (1987), Sagiya, Y. et al., Appl. Microbiol. Biotechnol., 42:358–363 (1994), Yamagata, H. et al., Proc. Natl. Acad. Sci. USA 86: 3589–3593 (1989)). However, it has recently been found that the rate of correct disulfide bond formation is not 100% in this expression system (Miyauchi, A. et al., J. Indust. Microbiol. Biotech., 21: 208–214, (1998)). The rate of correct disulfide bond formation seems to differ depending on the types of polypeptides. For example, the formation rate of a human epidermal growth factor is 80%.
To date, it is known that protein disulfide isomerase is involved in the intracellular formation of correct disulfide bonds of a protein. Particularly regarding yeast, there has been a report that co-expression of the gene of protein disulfide isomerase involved in disulfide bond formation and the gene of a protein having disulfide bonds has enabled production of proteins having correct disulfide bonds (Japanese Patent Application Laying-Open (Kokai) Nos. 6-38771 and 7-508881). Generally, in yeast, the eukaryote, disulfide bonds are formed while the protein is transferred from intracellular endoplasmic reticulum to the Golgi complex, and then secreted extracellularly after completion of the high order structure of the protein. As an example in bacteria, there has been a report that a target protein gene has been ligated to the gene of protein disulfide isomerase of molds and expressed in an expression system of bacteria of the genus Bacillus. In this example, protein disulfide isomerase is expressed as a fusion protein with the protein (Japanese Patent Application Laying-Open (Kokai) No. 11-75879).
An object of the present invention is to increase efficiency of the production of polypeptides with correct disulfide bonds in a bacterial expression system for genes encoding proteins where it is difficult or impossible for such bonds to form. This is done by allowing co-expression of a gene encoding a polypeptide having disulfide bonds and a gene encoding protein disulfide isomerase, so as to establish an environment wherein the polypeptide and protein disulfide isomerase co-exist. In this case, it is required to surmount problems of how an expression/secretion system should be established to allow co-expression of the gene encoding a polypeptide and the gene encoding protein disulfide isomerase within bacterial cells, to allow extracellular secretion of both the generated proteins, and to allow extracellular formation of correct disulfide bonds in the polypeptide.